Yang Yuanzhong, Boysen Reinhard I, Matyska Maria T, Pesek Joseph J, Hearn Milton T W
Australian Research Council Special Research Centre for Green Chemistry, Clayton, Victoria, 3800, Australia.
Anal Chem. 2007 Jul 1;79(13):4942-9. doi: 10.1021/ac0622633. Epub 2007 Jun 1.
In this study, the open-tubular electrochromatographic (OT-CEC) migration behavior of various peptides has been characterized using etched and chemically (n-octadecyl- and cholesterol-) modified capillaries, interfaced to an electrospray ionization mass spectrometer through a sheath liquid configuration. The stationary phases were fabricated by etching the inner wall of the fused-silica capillary and then chemically modifying the new surface through a silanization/hydrosilation reaction. Unlike some other OT-CEC stationary-phase preparation methods, leaching of the immobilized stationary phase and subsequent contamination of the electrospray ion source was largely avoided with this novel surface modification technology. The influence of the immobilized organic phases and those of the buffer electrolytes (pH, the type and content of organic solvent) on the retention and separation of the selected peptides was investigated. Significant peptide retention was found even at very low pH with both types of stationary phases, under conditions whereby the electrophoretic migration dominated the separation process. Due to the effective coverage of the etched surface by a silanization/hydrosilation reaction, adverse adsorption of charged analytes onto the capillary wall was minimized. As a result, very efficient and highly reproducible peptide separations were achieved over a broad pH range. Moreover, peptide-specific multizoning effects were observed. The origin of this novel phenomenon was explored. Compared to capillary electrophoresis electrospray ionization mass spectrometry system, much higher detection sensitivity could be obtained, since a larger amount of sample could be injected and stacked at the head of the open-tubular capillary column without deteriorating the separation performance. On the basis of these observations, these procedures have been adapted to allow the analysis of tryptic peptides generated from proteins.
在本研究中，使用蚀刻和化学（正十八烷基和胆固醇）修饰的毛细管对各种肽的开管电色谱（OT-CEC）迁移行为进行了表征，该毛细管通过鞘液配置与电喷雾电离质谱仪相连。固定相通过蚀刻熔融石英毛细管的内壁，然后通过硅烷化/硅氢化反应对新表面进行化学修饰来制备。与其他一些OT-CEC固定相制备方法不同，这种新颖的表面修饰技术在很大程度上避免了固定化固定相的浸出以及随后对电喷雾离子源的污染。研究了固定化有机相和缓冲电解质（pH值、有机溶剂的类型和含量）对所选肽的保留和分离的影响。在电泳迁移主导分离过程的条件下，即使在非常低的pH值下，两种类型的固定相都发现了显著的肽保留。由于硅烷化/硅氢化反应有效地覆盖了蚀刻表面，带电分析物在毛细管壁上的不利吸附被最小化。结果，在很宽的pH范围内实现了非常高效且高度可重复的肽分离。此外，还观察到了肽特异性的多区带效应。探索了这种新现象的起源。与毛细管电泳电喷雾电离质谱系统相比，可以获得更高的检测灵敏度，因为可以注入更多的样品并在开管毛细管柱的头部进行堆积，而不会降低分离性能。基于这些观察结果，这些方法已被调整以允许分析蛋白质产生的胰蛋白酶肽。
